Stempro media

3 d after EP, cells were resuspended in StemPro media (Gibco) containing StemPro Nutrients, low density lipoprotein 50 ng/ml, P/S 1%, glutamine 1%, Flt3 20 ng/ml, TPO 50 ng/ml, IL-6 50 ng/ml, IL-3 10 ng/ml, IL-2 10 ng/ml, IL-7 20 ng/ml, erythropoietin 3 ng/ml, GM-CSF 20 ng/ml, and SCF 100 ng/ml, and 2,000 cells/well were plated in a round bottom 96-well plate. In some wells, CSL362-ADC at a concentration of 10 ng/ml was added. After 14 d cells were collected, stained for CD33, GlyA/CD235a, CD14, CD15, and CD123, and acquired on a Fortessa.

Peripheral blood mononuclear cells (PBMCs) from 54 healthy adults (ranging in age from 18 to 70) were isolated from fresh heparinized peripheral blood, or from leukocyte reduction chambers, with Ficoll-Pague Plus (GE Healthcare) or Lymphoprep (StemCell Technologies). Leukocyte reduction chambers typically contained 1–2 × 10⁹ PBMCs. Four million total PBMCs were stained for flow cytometric analyses and the remaining cells were either immediately enriched for CD34⁺ hematopoietic progenitors using the EasySep Human CD34 Positive Selection Kit (StemCell Technologies) or were cryopreserved for later enrichment. Thawed CD34⁺ enriched HSPCs were cultured overnight in Stem Pro media (Invitrogen) supplemented with 100 U penicillin and 100 μg streptomycin and a cytokine cocktail including stem cell factor (SCF) (100 ng/ml), FLT3 ligand (50 ng/ml), and thrombopoietin (TPO) (10 ng/ml) (R&D Systems) to acclimate cells prior to sorting. CD34-enriched cells were then sorted using a FACS Jazz (BD Biosciences) for CD38⁻ CD45RA⁻ CD49f⁺ HSCs for in vitro experiments [54 ]. Post-sort analyses were typically > 98% HSCs.

PBMCs from a total of six chronic-phase CML patients (three of them do not have the BIM deletion polymorphism while the others do) were used. The presence of the BIM deletion polymorphism was detected using the method described previously [1 ]. PBMCs were thawed and allowed to recover overnight in serum-free StemPro media (Invitrogen, USA), supplemented with human growth factors [15 (link)], and 1X nutrient supplement (Invitrogen, USA) [34 (link)]. Cells were then subjected to drug treatment for 96 hours in the liquid media, harvested, washed, and seeded in methylcellulose (H4434; Stemcell Technologies, USA). The aim of this assay is to determine how the viability of the primary CML cells was affected during the 96-hour incubation with various drugs in the liquid media. Colonies were enumerated after 14 days.

CD34-enriched BP cells were thawed and allowed to recover overnight in serum-free StemPro media (Invitrogen), supplemented with human growth factors (GM-CSF 200 pg ml⁻¹, G-CSF 1 ng ml⁻¹, SCF 200 pg ml⁻¹, LIF 50 pg ml⁻¹, MIP-1α 200 pg ml⁻¹ and IL-6 1 ng ml⁻¹) and 1× nutrient supplement (Invitrogen). Cells were then subjected to drug treatment for 48 h, harvested, washed and seeded in methylcellulose (H4434, STEMCELL Technologies). Colonies were counted after two weeks.

Peripheral blood (PB) samples were obtained with appropriate consent and IRB approval from patients with CML at the University of California, Irvine (S. T. Ong, #IRB 01–59) and patients seen at the Singapore General Hospital (under SingHealth IRB approved protocols). Patient diagnosis and clinical responses are listed in the electronic supplementary material, table S1 [30 (link)]. PB mononuclear cells were obtained by centrifugation through Ficoll-Hypaque, washed in PBS and cryopreserved. CD34⁺ cells were selected by immunomagnetic beads (Miltenyi Biotech). To expand CD34⁺-enriched BP CML cells in vitro, cells were thawed and grown (overnight) in serum-free StemPro media (Invitrogen) supplemented with 1× nutrient supplements (Invitrogen) and a growth factor cocktail consisting of 200 pg ml⁻¹ granulocyte–macrophage colony-stimulating factor (GM-CSF), 1 ng ml⁻¹ granulocyte-colony-stimulating factor (G-CSF), 200 pg ml⁻¹ stem cell factor (SCF), 50 pg ml⁻¹ leukaemia inhibitory factor (LIF), 200 pg ml⁻¹ macrophage inflammatory protein-1 alpha (MIP-1α) and 1 ng ml⁻¹ interleukin 6 (IL-6). Cells were then subjected to drug treatment for 48 h in the same media. All cytokines were from PeproTech, except for GM-CSF (sargramostim, Immunex) and G-CSF (filgrastim, Amgen).