Methocult h4435 medium

Mononuclear cells were isolated from bone marrow samples on Histopaque density gradient separation (Sigma-Aldrich, St. Louis, MO). CD34+ cells were purified using immunomagnetic columns (Miltenyi Biotech, Paris, France). The purity of CD34+ cells, assayed by flow cytometry, varied between 90% and 95%. CFU-C assays (at day 0) were performed by plating 5,000 CD34+ cells on semisolid methylcellulose Methocult H4435 medium (StemCell Technologies, Vancouver, Canada). After 14 days of culture, hematopoietic colonies were enumerated, and a large fraction or the almost totality of CFU-Cs were plucked from methylcellulose and put (individually or by pools of 10 colonies) into RNA extraction buffer (Arcturus Bioscience Inc, Mountain View, CA). LTC-IC assays were carried out by seeding 40-60,000 CD34+ cells in MS5 stromal layers engineered to express HoxB4 protein, in order to amplify the number of leukemic LTC-IC, as previously described [19 (link)]. At week+5, cultures were sacrificed, and CFU-Cs originating from LTC-ICs were plucked, used for RNA extraction and analyzed for BCR-ABL1 mRNA expression.

The hematopoietic colony forming assay was performed in MethoCult H4435 medium (Stem Cell Tech) supplemented with Flt-3 L, IL-7, IL-3, SCF, and TPO (PeproTech, NJ, USA).

Transfected cells were resuspended in IMDM (Thermo Fisher Scientific, Waltham, Massachusetts, United States) supplemented with 2% FBS (Merck Group, Darmstadt, Germany) at a final concentration of 1 × 10⁴ cells/ml. For duplicate assays 250 µl of the cell suspension was added to 2.5 ml MethoCult^® H4435 medium (STEMCELL^™ Technologies), mixed thoroughly, and 2 × 1 ml of the cell suspension was dispensed into 35 mm cell culture dishes. The cells were incubated for 12 days at 37 °C. Colonies were enumerated based on their morphology. To assess serial replating capacity, quantified cultures were resuspended in IMDM/ 2% FBS, washed twice, and viable cells were quantified. Depending on the cell concentration and viability, 1000–2500 cells were again plated in MethoCult^®, as described above. The process was repeated up to four times, or until no colonies were detected.

The cultured CD34+ cells from all experimental and control groups were separated and centrifuged at 300×g for 5 min. One thousand cells were then seeded into 35-mm dishes (Nalge Nunc International) containing 0.5 ml of semisolid methylcellulose in Methocult H4435 medium (StemCell Technologies). The 35-mm dishes were incubated at 37 °C in a 5% CO₂ in air atmosphere for 14 days. Burst forming unit-erythroid (BFU-E), colony-forming unit-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte-macrophage (CFU-GM) colonies that were formed after 14 days were counted and classified based on morphology as described by the Atlas of Hematopoietic Colonies from Cord Blood. The CFU colonies were photographed using an inverted phase-contrast microscope (Nikon Instruments, Tokyo, Japan). CFU numbers were then calculated by dividing the number of colonies at day 14 by the number of cells plated and multiplying this value by 10,000 which reflected the colony-forming ability of 10,000 cells.

Sorted CD45 + cells were cultured with Methocult H4435 medium (STEMCELL Technologies Inc, cat. #04435, Vancouver, BC, Canada) for 14 days. The formed colonies were counted and classified based on their morphology. The quantification analysis was normalized to the initial number of seeded leukemic or CB cells (20,000 leukemic cells and 500 cells of CB CD34 +) and compared to the corresponding HD CFU results.