Myelocult h5100 medium

To mimic the AML-ME and investigate a crosstalk between BM-derived adipocyte and/or osteocyte subpopulations and leukemic cells, a “patient-in-a-dish” ex-vivo system was generated. Leukemic cells were sorted using FACS (Aria II; BD Biosciences) based on the leukemia-associated immunophenotype (LAIP), specific for each patient. The MNCs isolated from whole cord blood (CB) samples were employed as a control and were further enriched for CD34 + progenitor cells, using magnetic CD34 MicroBead Kit (human; Miltenyi Biotec, cat. #130,046–702, Bergisch Gladbach, Germany).
AML- or HD-induced adipocytes/osteocytes were then cultured in the MEM-α. CD34 + cells derived from either leukemic or CB cells were co-cultured (for 10 days) with the above MSC feeder layer at a density of 2 × 10⁴ cells/cm² in the MyeloCult™ H5100 medium (STEMCELL Technologies Inc, cat. #H5100) with the addition of 1% penicillin–streptomycin solution. Cells were trypsinized, harvested and sorted for CD45 + cells, using CD45 Microbeads (human; Miltenyi Biotec, cat. #130–045-801) for simultaneous conduction of the colony forming unit (CFU) assay. The cell viability was assessed using the flow cytometry analysis.

BM-MSCs were exposed to pesticides only during the 21 days of expansion culture before co-cultures with normal CD34⁺ cells, and CAFC assays were performed as previously described and as detailed in supplementary methods [22 (link)]. Briefly, CD34⁺ cells were thus co-cultured with the pesticide-exposed BM-MSCs for 7 days (37 °C, 5% CO₂), seeded at 6000 CD34⁺/cm² (ratio CD34⁺ cells/BM-MSCs = 1/3) in Myelocult™ H5100 medium (StemCell Technologies, Vancouver, Canada). At the end of coculture, CD34⁺ cells were sorted (BD FACSMelody^TM cell sorter, Becton-Dickinson) and cultured on MS5 stromal cells [25 (link)] for 5 additional weeks in 96-well plates (37 °C, 5% CO₂) at 6 different dilutions (range 1–100 CD34⁺ cells/well) in 0.2 mL of Myelocult™ H5100 medium. After 5 weeks of co-culture, each well was examined by inverted phase contrast microscopy (DMI 3000 B, Leica) to identify cobblestone areas. The percentage of wells with at least one CAFC was quantified and the frequency of CAFC in the initial CD34⁺ cell population was calculated as previously described [26 (link)]. The CAFC proliferative capacity was determined by subculturing the wells containing one cobblestone area in semi-solid medium (MethoCult^TM H4434, StemCell Technologies) to determine the mean total number of clonogenic progenitors (including CFU-GM, BFU-E and CFU-M) generated by each CAFC.

Fluorescence activated cell sorted subsets were seeded in up to four replicates in 96‐well collagen‐coated plates and cultured on irradiated (80 Gy) M2‐10B4 stromal feeder cells (American Type Culture Collection [ATCC], Manassas, VA, USA) in MyeloCult H5100 medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with penicillin‐streptomycin (Gibco^®, Thermo Fischer Scientific Inc., Waltham, MA, USA) and 10⁻⁶ mol/l hydrocortisone 21‐hemisuccinate (StemCell Technologies) with weekly half‐change of medium. After 6 weeks of co‐culture, cells were harvested and transferred to MethoCult H4435 (StemCell Technologies) for an additional 2 weeks. CFCs were counted and scored for multi‐lineage (colony‐forming unit‐granulocyte/erythroid/macrophage/megakaryocyte (CFU‐GEMM)); erythroid (burst‐forming unit ‐ erythrocyte (BFU‐E)) and myeloid morphology [colony‐forming unit ‐ granulocyte (CFU‐G), colony‐forming unit ‐ macrophage (CFU‐M) and colony‐forming unit ‐ granulocyte/macrophage (CFU‐GM)]. Ten colonies from each subset of the originally seeded cells were individually picked and cytospun onto poly‐L‐lysine coated slides (Thermo Fischer Scientific Inc., Waltham, MA, USA) and allowed to air‐dry for subsequent FISH analyses.